Herpesviruses are omnipresent human pathogens that cause a number of important diseases. Devising better strategies to combat these viruses requires the in-depth understanding of how they replicate. This proposal focuses on nuclear egress during which nucleocapsids bud into the inner nuclear membrane, a critical step in the assembly and release of progeny virions. The viral nuclear egress complex (NEC) is the key player in this process, but the detailed mechanistic knowledge of its function is lacking. The long-term goal of this research is to elucidate the atomic-level mechanism of herpesvirus nuclear egress, to improve the fundamental knowledge of this process and to identify and characterize novel antiviral targets. The objective of this proposal is to fully explore the mechanism of NEC-mediated membrane deformation during nuclear egress by using an amalgamation of structural, biophysical, and cell-based strategies. This proposal is driven by the central hypothesis, based on substantial preliminary data, that the NEC alone mediates membrane budding and, possibly, scission in oligomerization-dependent manner. Aim 1 will focus on determining the mechanism of membrane deformation by the HSV-1 NEC by using the in vitro membrane budding assay with giant unilamellar vesicles (GUVs) in combination with light microscopy and cryoelectron microscopy. The membrane budding assay will also be used to reconstitute capsid budding in vitro. The goal of Aim 2 is determine the structure of the HSV-1 NEC and to map functionally important regions by structure- guided mutagenesis in combination with in vitro assays and the in vivo characterization of recombinant viruses. Aim 3 will concentrate on revealing the conserved and the virus-specific features of the NEC mechanism in alpha and gammaherpesviruses by extending the biochemical and the structural studies to NECs from pseudorabies, Epstein-Barr, and Kaposi's Sarcoma herpesviruses. The proposed work will leverage the combined expertise in biochemistry and structural biology (Heldwein lab) with expertise in virus mutagenesis and live-cell fluorescent imaging (Smith lab). The outcome of these studies will be the structure of the NEC and the mechanism by which it enables membrane deformation during nuclear egress. Obtaining the crystal structure of the NEC will have a major impact in the field of herpes virology by yielding mechanistic and functional insights unavailable by any other approaches. Furthermore, detailed in vitro studies of the NEC-mediated membrane budding may uncover a novel membrane budding mechanism. Importantly, by correlating the in vitro properties of the NEC mutants with the in vivo phenotypes of the corresponding recombinant viruses, the NEC structure and the observations from in vitro budding models will be directly related to the nuclear egress in infected cells. A detailed knowledge of the structural mechanism of membrane remodeling by the NEC during nuclear egress will benefit not just the herpesvirus field, but also significantly advance the current knowledge of cellular processes.